Trans mono-unsaturated fatty acids and tissue factor activity in a monocytic cell line

Abstract

Tissue factor is the initiator of the coagulation cascade [l], a membrane associated protein requiring phosphatidylserine and Ca2' cofactors for full activity. Upon presentation at the site of injury, TF converts factor-Vll to factor Vlla and forms a membrane associated TFNlla complex. This in turn modifies transiently binding factor X to Xa, which is then released to propogate the coagulation cascade.Phosphatidylserine has associated fatty acids which may modulate the activity of TF. Other authors have shown effects on TF activity by incorporation of different fatty acids into TF-expressing cells. n-3 fatty acids reduce TF acitivity significantly in human monocytes after 12 weeks of dietary supplementation [2]. Adrenic acid (22:4,n-6)-enriched human umbilical vein endothelial cell cultures have showed an increase in TF activity after thrombin stimulation [3]. Lipoproteins can also modulate TF activity, either increasing or decreasing activity depending on the lipoprotein and its oxidative state [4].There has been no information on the effects of trans fatty acids on TF activity. The aim of this study was to evaluate the effects of cis and trans monoenoic fatty acids on TF activity in a human monocytic cell line.THP-1 monocytic cells were cultured with oleic acid (cis 18:1A9) or elaidic acid (trans 18:1A9) bound to BSA at 35pM fatty acid final concentraton in RPMl 1640 with 10% foetal calf serum. The cells were incubated in this culture media with the fatty acids for 48h and then transferred into SFM which was also fatty acid-BSA supplemented. The cells were then exposed to PMA for 16h to initiate expression of TF activity. The control cells were treated with fatty-acid free BSA. After 16h the cells were harvested, washed and the TF activity measured by a one stage clotting assay [4]. The fatty acid content of the cells was analysed by gas chromatography .The results indicate an increase in TF activity in response to increasing PMA concentrations with maximal activity at 10-1000nM: above this concentation PMA becomes toxic. The supplemented cells have increased TF activity over controls wheras trans supplemented cells had a higher TF activity than controls. The percentage of oleic acid in the cellular lipids was increased from 17% to 47% and from less than 1 % to 21 % in the case of elaidic acid.The level of fatty acid incorporation was considered rather high and the incubation times with fatty acids were reduced to 24h to yeild an incorporation of 7% above baseline levels. Time course experiments were then conducted following incubation with fatty acids (and fatty acid free controls) followed by stimulation by PMA at IOnM for 2h. After this, the cells were given three washes in PBS and resuspended in SFM with 35pM fatty acid BSA. Samples of cells were then taken at intervals up to 13h washed and TF activity measured via the a stage clotting assay. TF = Tissue factor PBS = Phosphate buffered saline PMA = Phorbol myristoyl acetate BSA = Bovine serum albumin SFM = serum free media 3 1 2 \ \ 1 0 ' 0 2 4 6 8 1 0 1 2 ...